The present invention relates generally to genetic engineering and more specifically to use of recombinant DNA techniques to secure expression of protein synthesis in selected unicellular organisms.
For many years, microorganisms have provided a source of commercially significant products such as antibiotics, enzymes and other biologically active proteins, alcohols and the like. Bacteria and yeasts have been the subject of intensive research effort which has had as its goal the enhancement of the cellular and extracellular yield of such commercial products by naturally-occurring and artificially mutated (e.g., radiation treated) strains of organisms. While manipulation of fermentation and product harvesting process parameters can frequently bring about dramatic increases in product yield for selected strains, it is most frequently the case that no degree of modification of culture and isolation conditions will result in enhancing an organism's "expression" of a particular product as evidenced by increased product yields.
A focus of genetic engineering in the recent past has been the use of recombinant DNA methodologies for the purification and amplification of genetic material. U.S. Pat. No. 4,237,224 to Cohen, et al., for example, relates to transformation of procaryotic unicellular host organisms with "hybrid" viral or circular plasmid DNA which includes exogenous DNA sequences. The procedures of the Cohen, et al. patent first involve manufacture of a transformation vector by enzymatically cleaving viral or circular plasmid DNA to form linear DNA strands. Selected foreign DNA strands are also prepared in linear form through use of similar enzymes. The linear viral or plasmid DNA is incubated with the foreign DNA in the presence of ligating enzymes capable of effecting a restoration process, and "hybrids" are formed which include the selected foreign DNA segment "spliced" into the viral or circular DNA plasmid. Transformation of compatible host unicellular organisms with the hybrid vector and propagation of transformant cells results in the formation of multiple copies of the foreign DNA in the host cell population. In some instances, the desired result is simply the amplification of the foreign DNA and the "product" harvested is DNA. More frequently, the goal of transformation is the expression by the host cells of the foreign DNA in the form of large scale synthesis of isolatable quantities of, e.g., commercially significant protein and polypeptide fragments coded for by the foreign DNA.
The success of procedures such as described by Cohen, et al. is due in large part to the ready availability of restriction endonuclease enzymes which facilitate the site-specific cleavage of both the unhybridized DNA vector and, e.g., eukaryotic DNA strands containing the foreign sequences of interest. Cleavage in a manner providing for the formation of complementary "ends" on the linear DNA strands greatly enhances the likelihood of functional incorporation of the foreign DNA into the reconstituted vector formed by ligating enzyme treatment. Verification of hybrid formation is facilitated by chromatographic techniques which can, for example, distinguish hybrid plasmids from non-hybrids on the basis of molecular weight. Other useful verification techniques involve radioactive DNA hybridization.
Another manipulative "tool" largely responsible for successes in transformation of procaryotic cells is the use of selectable "marker" gene sequences. Briefly put, hybrid vectors are employed which contain, in addition to the desired foreign DNA, one or more DNA sequences which code for expression of a phenotypic trait capable of distinguishing transformed from non-transformed host cells. Typical marker gene sequences are those which allow a transformed procaryotic cell to survive and propagate in a culture medium containing metals, antibiotics, and like components which would kill or severely inhibit propagation of non-transformed host cells.
Reports abound concerning successful transformation of host microorganisms such as Escherichia coli with common circular DNA plasmids such as pBR322 which has been hybridized to incorporate exogenous procaryotic or eukaryotic genes. Relatively large quantities of the protein products coded for by the foreign genes can often be isolated from the transformant cells or from the culture medium in which the cells are propagated.
Rather expectedly, the literature provides few details of the numerous unsuccessful attempts at securing desired expression of foreign gene directed protein synthesis using standard host/vector methodologies. While in some instances transformation "failures" are simply the result of defective hybrid formation, it is nonetheless frequently the case that an "unsuccessful" vector is verified as including all portions of a foreign gene sequence that are otherwise necessary for directing synthesis of the desired protein product. Similarly, the lack of success in some experiments can be attributed to a generalized incompatibility between host and vector. In many cases, however, stable incorporation of the vector (accompanied by extensive replication and expression of marker gene phenotype) does take place, but expression of desired protein synthesis does not.
The failure of host microorganisms to express proteins coded for by foreign DNA sequences may also be a problem in recombinant DNA methodologies involving direct incorporation of exogenous DNA into chromosomes of microorganisms. While chromatographic and radioisotopic DNA hybridization studies might verify incorporation of entire genes (including necessary promoter and leader sequences) into host genomes, there may be no corresponding expression of the protein coded for by the gene.
Among the hypotheses advanced in explanation for the failure of otherwise fully operational transformation systems to provide the desired yields of exogenous gene directed protein are: (1) interference by host cell constituents in transcription and/or translation of the foreign DNA; and (2) interference by host cell constituents in the accumulation of readily isolatable quantities of gene products in the host cells.
As indicated by the above, therefore, there exists a general need in the art for methods and materials which will serve to enhance the expression of endogenous gene directed protein synthesis in naturally occurring and mutant microorganisms as well as expression of exogenous gene directed protein synthesis in microorganisms which have been the subject of transformations with exogenous DNA by recombinant DNA methodologies.